Invasive adherence to host tissues by bacterial pathogens is often facilitated by means of elongated hairlike proteinaceous fibers called pili or fimbriae that protrude outwardly from the microbial cell surface. In Gram-negative pathogenic bacteria the role of pili as colonization agents in pathogenesis is well recognized and the overall mechanism of pilus assembly is clearly defined from over fifty years of research. The most structurally characterized Gram-negative pili are the type I form, found, for example, in the enteropathogenic E. coli, and type IV form, found, for example, in species of Neisseria and Pseudomonas as well as in E. coli. Typically, the Gram-negative pili are long (1 to 4 μm in length) and thin (5 to 8 nm in width), and also display both flexible and robust structural properties. These pili are generally comprised of a series of non-covalently linked multiple protein subunits whose assembly is dependent upon specific chaperone proteins, but independent of any enzymatic activity. Frequently, a protein with adhesive properties is positioned at the tip of the pili. It is generally considered that the intervening length of protein subunits from the microbial surface promotes an unhindered contact between the adhesive tip protein and corresponding host cell receptor sites, which are potentially represented by components of the extracellular matrix (ECM) or specific carbohydrate moieties of glycoproteins and glycolipids (Scott J. R. and Zähner D, 2006, Mol Microbiol 62, 320-330; Telford, J. L., et al. 2006, Nat Rev Microbiol 4, 509-519).
The presence of Gram-positive pilus-like structures was actually first observed in the late 1960's by electron microscopy of Corynebacterium renale (Yanagawa, R. et al. 1968, Jpn J Vet Res 16, 31-37), and in the subsequent years pili have been found in several other Gram-positive bacterial species, including the very recent discovery of pili in the three main invasive disease-causing streptococcal pathogens in humans, i.e., Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae (Telford, J. L., et al. 2006, Nat Rev Microbiol 4, 509-519). The most detailed characterization studies of Gram-positive pili originate from the corynebacteria, streptococci, and bacilli pathogens.
Unlike in the Gram-negative bacteria, the pili in Gram-positive bacteria are much thinner in width (2 to 3 nm) and more difficult to visibly distinguish which also suggests why the presence of these pili may have been over-looked in many species of Gram-positive bacteria (Kang, H. J. et al. 2007, Science 318, 1625-1628). To date, the most thorough description of the pilus-assembly process, that is also generally representative of all Gram-positive pili, has been carried by in vivo characterization studies of pili biogenesis in Corynebacterium diphtheriae (Ton-That, H. and Schneewind, O. 2004, Trends Microbiol 12, 228-234). Structurally, the prototype pili appear as polymers composed of covalently cross-linked protein subunits (called pilins) that are also covalently anchored at the base to the peptidoglycan component of the cell wall, with both of these covalent bonds being enzymatically dependent upon catalysis by different sortase family membrane-bound transpeptidases, i.e., the pilin-specific and the housekeeping sortases, respectively (Mandlik, A. et al. 2008, Trends Microbiol 16, 33-40). The Gram-positive pilus is typically composed of three pilin subunits and, in the case of C. diphtheriae, the genes named as SpaA (sortase-mediated pilin assembly) for the major pilin subunit that exclusively forms the shaft or backbone of the pilus, SpaB for an ancillary minor pilin subunit, and SpaC for another minor pilin subunit with adhesive properties located at the tip of the pilus (FIG. 1). The genes encoding these three pilin subunits are localized within the same loci as a pilin gene cluster along with at least one gene encoding a pilin-specific sortase in close proximity. As well, the genes within the pilin cluster are frequently flanked on both ends by transposable elements suggesting an origin by horizontal gene transfer. The transcription of all these genes is in the same direction and indicative of operon regulatory control (Scott J. R. and Zähner D, 2006, Mol Microbiol 62, 320-330).
The revised model of the overall Gram-positive pilus assembly process, which is dependent upon several different conserved motifs and domains within the primary sequence of each pilin subunit, includes four basic stages (Mandlik, A. et al. 2008, Proc Natl Acad Sci USA 105, 14147-14152; Telford, J. L., et al. 2006, Nat Rev Microbiol 4, 509-519)-(FIG. 1). In the first stage, the pilin proteins, each of which contain a N-terminal signal peptide, are secreted through the bacterial cell membrane by the Sec-dependent pathway and then retained in the cell membrane by the presence of a C-terminal membrane-spanning domain consisting of a hydrophobic region of about 20 residues and a positively charged tail.
In the second stage of the assembly process, the cell wall sorting signal (CWSS), preferably the LPXTG-motif, which also immediately precedes the membrane-spanning domain, becomes available for sortase-dependent cleavage of the cell membrane-anchored pilin proteins. The pilin-specific sortase cleaves this five residue motif between the threonine (T) and glycine (G) residues and forms an acyl-enzyme intermediate involving a covalent thioester bond between the carboxyl group of the threonine residue and a cysteinyl thiol found within the catalytic pocket of the sortase.
The third stage represents the polymerization of the pilin subunits by isopeptide bond formation and involves the cleavage of the thioester bond and the release of the sortase from the pilin subunit by the nucleophilic attack of the ε-amino group from the side chain of a lysine (K) residue conserved in the pilin-motif (WXXXVXVYPKN) of a second pilin subunit. An amide bond is thought to form between the C-terminal carboxyl of the threonine residue in the first pilin subunit and the side chain amino group of the pilin-motif lysine from a second pilin subunit still bound as a covalent thioester with an another pilin-specific sortase (Budzik, J. M. et al. 2008, Proc Natl Acad Sci USA 105, 10215-10220). In this model of pilus assembly, the growing polymeric structure is fed by additional pilin subunits at the base of the pilus and the overall length governed by the amount of available pilin subunits associated with pilin-specific sortases. Since the pilin-motif is a characteristic feature of the major (SpaA) and ancillary minor (SpaB) pilin subunits, but missing in the primary sequence of the minor pilin subunits (SpaC) displaying adhesive properties, this pilin subunit is likely located at the tip of the pilus shaft and the first pilin subunit to initiate pilus polymerization.
The attachment of the polymerized pilus to the cell wall represents the fourth stage of the assembly process. Herein, the ancillary minor pilin subunit (SpaB) signals the cessation of pilus polymerization, but only when presented in association with a housekeeping sortase, whose gene is encoded somewhere else on the genome. In this final stage, the growing polymeric structure of major pilin subunits (SpaA) is transferred from a thioester linkage with a pilin-specific sortase to form an amide bond with the side chain of the lysine in the pilin-motif of SpaB minor pilin subunit, which is coupled as a housekeeping sortase acyl-enzyme intermediate. The nucleophilic attack by the amino group of the pentapeptide of the peptidoglycan lipid II precursor then permits the housekeeping sortase to catalyze the attachment of the SpaB pilin-linked pilus polymer to the cell wall. The E-box represents a third and less characterized conserved primary sequence motif (YXLXETXAPXGY) found between the LPXTG- and pilin-motifs of the pilin subunits from many Gram-positive bacteria.
Thus far, three-dimensional (3-D) structure determinations by x-ray crystallography have revealed structural insights into the assembly and function for only two Gram-positive pilin subunit proteins. Krishnan et al. (2007, Structure 15:893-903) had solved the crystal structure for the minor pilin GBS52 of Streptococcus agalactiae and revealed the presence of two IgG-like domain folds that share a structural similarity with the S. aureus collagen-binding protein Cna which also indicates how this minor pilin subunit could facilitate pilus adherence to a specific host tissue. The crystal structure of the major pilin Spy0128 from Streptococcus pyogenes, solved by Kang et al. (2007, Science 318, 1625-1628), had demonstrated how self-generated intramolecular isopeptide bonds between the side chains of lysine and asparagine residues within the pilin subunit could also complement the sortase-catalyzed intermolecular isopeptide bonds for maintaining the overall strength and stability of pili.
The majority of probiotic microbes are members of the Gram-positive lactobacilli and bifidobacteria and have a long tradition of use in fermented foods and dairy products (Goldin, B. R. and Gorbach, S. L. 2008, Clin Infect Dis 46, S96-S100; Ljungh, A. and Wadstrom, T. 2006, Curr Issues Intest Microbiol 7, 73-89; Salminen, S. et al. 1998, Br J Nutr 80, S147-S171). Pilus structures of probiotic lactobacilli or genes encoding these pilus structures have not been described in the literature. The presence of pilus-like structures or polynucleotides has never been shown in Lactobacillus rhamnosus. 